Embryonic stem cells (ESC) will revolutionize medical research and patient treatment. However, two major hurdles do not allow the rapid transition of ESC therapies to clinical settings: 1) the lack of protocols precluding homogenous cell populations that exactly reproduce those in vivo; 2) the uncertainty about the fate stability of ESC-derived cells after in vivo grafting. Overcoming these limitations will ameliorate safety concerns associated with ESC-derived cell therapies. Our long term goal is to efficiently generate ESC-derived cells that functionally integrate into organs. We have recently developed efficient protocols to derive terminal cell fates from ESC. When expressed in differentiating ESC, Ngn2- Isl1-Lhx3 (the NIL transcription factors) and Ngn2- Isl1-Phox2a (the NIP transcription factors) are sufficient to program spinal or cranial motor neuron identity respectively. This also happens extremely rapidly: Within 48 hours more than 97% of the cells acquire all the features of terminally differentiated motor neurons. Moreover, programmed neurons correctly integrate into the developing spinal cord projecting axons mirroring the endogenous neurons. Although, NIL and NIP factors do not provide the motor neuron subtype identity required for precise muscle innervation, our preliminary data suggests that it can be acquired by the activity of developmentally relevant signals. We hypothesize that NIL and NIP factors program generic motor neuron fate through a rapid transcriptional sequence, and that subtype identity can be independently imposed either by genetic factors or by the host environment after grafting. Here we propose 3 aims to test these ideas: Aim 1- To understand the molecular mechanisms of direct cell programming, we will map the NIL and NIP genetic and epigenetic requirements for efficient programming. This knowledge will facilitate the future design of programming strategies for different clinically relevant cell types. Aim 2- To increase the cellular precision of direct programming, we will impose subtype identity to NIL- programmed neurons by the activity of developmentally relevant signals or transcription factors. This novel strategy will generate neurons at a level of efficiency and precision compatible with clinical applications. Aim 3- To dissect the influence of the host tissue on ESC-derived neurons, we will test the cellular stability of NIL-programmed neurons after implantation into the spinal cord. These results will investigate if ESC-derived neurons change fate after interacting with the host tissue. Completing this proposal will impact not only future therapies for spinal cord injurie, but will also produce general principles to differentiate disease relevant cells at high efficiency These are necessary steps to accelerate the transition of ESC to clinical applications.